Bovine milk has an average protein content of 35 mg/mL. The two major protein fractions in bovine milk are the caseins and the serum milk proteins (frequently referred to as whey proteins), which account for approximately 80% and 20% of total protein, respectively.
Casein is a mixed complex of phosphoproteins that are present in all mammalian milk as colloidally dispersed micelles 50 to 600 nm in diameter. The assembly of casein micelles probably proceeds via two routes; hydrophobic interaction between groups on different molecules and calcium phosphate nanoclusters acting as a neutralizing bridge between two negatively charged phosphoseryl clusters. Small amounts of calcium phosphate together with serum ionic calcium play a significant role in micellar structure.
Casein can be separated from the whey proteins of milk by gel-filtration, high-speed centrifugation, salting-out with appropriate concentrations of neutral salts, acid precipitation at pH 4.3-4.6, precipitation with ethanol, precipitation with an anionic polysaccharide, and coagulation with rennet or other proteolytic enzymes. The majority of methods are impractical for commercial use. Commercially, for economic reasons, casein is separated from whey proteins by acid or rennet coagulation.
β-Casein is one of the four main casein proteins in milk. It has excellent functional properties, such as foaming and emulsification, and is suitable for use in a variety of food preparations. β-Casein is also the source of numerous peptides with biological/physiological activity, for example peptides that have anti-hypertensive activity, or promote mineral absorption.
In recent years, membrane technology (principally microfiltration) has also been used for the separation of casein from whey protein in milk. A few processes have been developed for the separation of casein from whey protein in milk using inorganic, primarily ceramic, microfiltration (MF) membranes (see, e.g., U.S. Pat. No. 6,485,762, incorporated herein by reference in its entirety). These processes are typically performed at a temperature in the range 45-55° C. (see, e.g., U.S. Pat. No. 6,623,871). Processing of milk at temperatures in this range can cause problems with growth of thermophilic bacteria and associated biofouling problems, fouling caused by calcium phosphate formation and deposition, enzyme activity resulting in reduced levels of intact casein and/or whey proteins, protein aggregation/gelation, and shortening of membrane life.
U.S. Pat. No. 5,169,666 discloses an ultrafiltration process for producing “humanized” bovine milk with casein/whey ratio of permeate of about 40:60. The disclosed microporous low temperature ultrafiltration process is based on 0.1-0.2 μm membranes in combination with pH and salt precipitation. The resulting permeate yields approximately 60% β-casein relative to the casein fraction.
Approximately 95% of total casein in milk is present in the form of micelles. The remainder is present in the serum phase of milk, with β-casein being the principal component of serum casein. β-Casein is the most amphiphatic and proline-rich of all the caseins with a strong potential for hydrophobic interactions. The strength of hydrophobic interactions decreases with decreasing temperature, which results in β-casein being monomeric in solution at temperatures between 0° C. and 4° C. On lowering the temperature of skim milk, the maximum concentration of β-casein in the serum phase of the milk is at temperatures in the range 0-5° C. Increasing the length of time that skim milk is stored at low temperature also increases the quantity of β-casein liberated into the serum phase.
In aqueous solution, β-casein has the ability to self-associate and form micelles with increasing temperature, in a process driven and stabilized by hydrophobic interactions. Micelles of β-casein have a diameter of approximately 30-34 nm, with about 15 to 50 monomers of β-casein per micelle. The formation of micelles by β-casein is strongly dependent on the concentration of β-casein, temperature, concentration of calcium, pH and ionic strength. The critical micelle concentration (concentration of β-casein below which formation of micelles will not occur) is reported to be 0.05 g/L at 40° C. With increasing temperature, the size of β-casein micelles increases in the temperature range 0 to 20° C., and the size of β-casein micelles remains constant irrespective of any further increase in temperature greater than 20° C.
Caseins exhibit susceptibility to charge neutralization, association and precipitation, due to the binding of calcium ions to phosphoseryl clusters on the molecule. However, the binding of calcium ions to β-casein molecules is very dependent on temperature. At 37° C., calcium concentrations in the range of 8-15 mM are sufficient to induce precipitation of β-casein. If the temperature is lowered to 1° C., β-casein remains soluble at calcium concentrations up to 400 mM.
Protein is the principal structural element of cheese. The two major components of the protein fraction in cheese are αs1- and β-casein. αs1-Casein is extensively hydrolyzed (up to about 80% hydrolysis) during ripening of Cheddar cheese, with the rate of hydrolysis being most rapid during the first 30-60 days of ripening. Conversely, only about 20-30% of total β-casein is hydrolyzed in Cheddar cheese during ripening, with the rate of hydrolysis being essentially constant during ripening.
The ratio of αs1-:β-casein in milk influences its rennet coagulation properties. Hydrolysis of β-casein enhances the heat-induced functional properties, especially meltability of cheese. Thus, reducing the concentration of β-casein in milk (i.e., increased αs1-:β-casein ratio) improves the functional properties of cheese, particularly its meltability.
Despite the apparent desirability of removing some β-casein from milk both to enhance certain properties of cheese and to obtain a natural emulsifier for use in other products, no commercially viable method of removing functional β-casein from milk has been developed. The existing methods result in contamination of milk and β-casein by enzymes and other impurities (products of β-casein hydrolysis), and generate large amounts of waste sludge that has no commercial value.
There is thus a need in the dairy industry for a process that can be used for purification of β-casein and its efficient removal from milk. The β-casein purification scheme should be more efficient and less expensive than classic purification protocols using ceramic membranes, which represent the current industry standard.